Summary of Work: The physiological mechanisms associated with neurogenesis and gliogenesis in the embryonic mammalian CNS have become an area of increasing study. Here, they are being studied using a novel strategy combining surface phenotyping with flow cytometry to access directly the physiological properties emerging during neuronal and glial cell lineage progression. Evidence has accumulated that neurotransmitters like acetylcholine (ACh) and GABA appear during neurogenesis where they are thought to play key roles in this process. During FY2000 neural stem cells and neuronal and glial progenitors were isolated from the embryonic cortex by phenotyping and flow cytometry. Stem cells either self-renewed or differentiated into neurons or glia depending on specific growth factors added to the culture. Clonal analysis showed that over time in culture cells underwent self-regulated lineage progression in the undisturbed microenvironment. Proliferating precursors were contiguous with proliferating progenitors, which were arrayed adjacent to differentiating neurons, astrocytes or oligodendrocytes depending on the lineage. This shows that in vitro all of the developmental signals necessary to generate the different stages of neuronal and glial lineages are conveyed in the microenvironment among the cells. Physiological properties and specific neurotransmitter receptor ion channel functions emerging de novo during cell lineage progression recapitulated those appearing in vivo. ACh regulated neuronal progenitor cell but not stem cell proliferation both in vitro and in vivo. Pharmacological block of cholinergic signaling at muscarinic receptors prevented cells from re-entering the cell-cycle and synthesizing DNA. Instead, the cells underwent apoptosis and died. Thus, ACh plays a key role in cortical progenitor cell expansion. Other research has shown that GABA mediates mitogenic, motogenic and morphogenic roles during cortical neurogenesis. GABA stimulated 1) progenitor cell proliferation acting via GABA(A) receptors, 2) postmitotic neuron migration via GABA(B) and 3) neurite outgrowth of postmigratory cortical neurons via GABA(A) receptors. ACh did not share these latter roles. The morphogenic effects of GABA resulted from constitutive synthesis and release from a surface-accessible compartment. This morphogenic role of GABA disappeared following neurite outgrowth and the formation of functional synapses. In summary, the results show that in the rat two widely distributed neurotransmitters play important mitogenic and/or motogenic and morphogenic roles during neurogenesis before they mediate brief synaptic signals.